Human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome (AIDS) continues to present a formidable challenge to health in developing countries. In the Western world, therapeutic strategies that target HIV-1 replication and maturation have had a prominent impact on disease progression. The high cost of current treatment, high toxicity of the drugs and lack of cure, however, means that the development of safe and effective vaccines remains paramount for control of the AIDS pandemic.
HIV-1 is a complex retrovirus encoding six regulatory and auxiliary genes not found in the simple retroviruses, namely, tat, rev, nef, vif, vpr and vpu (Table 1). In eukaryotic cells, only completely spliced mRNAs are exported to the cytoplasm for translation. Unspliced or partially spliced RNAs are retained and eventually degraded in the nucleus. In this way, proteins encoded by the tat, rev and nef genes (designated Tat, Rev and Nef) derived from multiply spliced RNA species, are expressed first and constitute early HIV-1 gene expression. In order to express singly spliced RNAs and also to transport the full length unspliced RNA genome into the cytoplasm for packaging, HIV-1 has developed means to overcome the restrictions on RNA transport. The regulatory proteins, Tat and Rev, are essential for HIV-1 replication since mutations in these proteins eliminate HIV-1 production (Dayton A. I., et.al. (1986) Cell, 44:941–947, and Fisher, A. G., et.al. (1986) Nature, 320:367–371.).
The auxiliary genes are derived from exons positioned entirely upstream of the HIV-1 envelope gene e.g. vif or exons upstream of as well as within env but in different reading frames e.g. tat, rev. The efficiency of splicing in part regulates the levels of gene expression of the different auxiliary proteins.
Following integration of HIV-1 proviral DNA, predominantly truncated forms of mRNA are synthesised by the cellular RNA polymerase II which interacts with sites on the 5′ long terminal repeat (LTR) of the proviral DNA. tat is one of the first genes to be expressed and carries a nuclear localisation signal. It is a potent transcriptional activator that enhances LTR-directed transcription up to a thousand fold. Continuous expression of Tat ensures a positive feed back loop for continued high level gene expression. Unlike conventional transcriptional activators that interact with DNA sequences, Tat binds directly to the 5′ ends of all HIV-1 RNAs at a specific stem-loop secondary structure, TAR (transactivating response element). The structure of the loop is highly conserved and essential for Tat function. The structure of the Tat/TAR interaction has been analysed using nuclear magnetic resonance (NMR) (Puglisi et al. (1992) Science, 257:76–80). Tat binds TAR in association with the cellular protein, cyclin T, which in turn binds CDK9 that phosphorylates the RNA polymerase II C-terminal domain, thereby promoting the elongation of RNA transcripts. These Tat cellular cofactors are only present in activated cells, their absence represses transcription of proviral DNA resulting in a ‘quasi latency’ in T lymphocytes. Tat is expressed from two exons, both the nuclear localisation signal and the TAR binding region are located in the first exon. Tat is secreted from infected cells and can exert heterologous effects on neighbouring cells. These include cellular activation (Hofman et al. (1993) Blood, 82:2774–2780.), induction of cellular apoptosis (Macho et al. (1999) Oncogene, 18:7543–7551.1999) functioning as a secretable growth factor (Trinh, D. P. et.al. (1999) Biochem. Biophys. Res. Commun., 256:299–306.) and modulating host cell protein synthesis in favour of viral protein synthesis (Xiao et al. (1998) Biochem. Biophys. Res. Commun., 244:384–389.1998). Therapeutic strategies targeting Tat will therefore have a strong impact on HIV-1 infection.
The small multiply spliced mRNAs encoding Tat, Rev and Nef predominate during early phase after infection. When a threshold level of Rev is produced, unspliced and singly spliced RNAs accumulate in the cytoplasm for translation, allowing productive infection to proceed. Failure to generate this threshold level of Rev may contribute to HIV quasi latency. Rev can only bind to RNAs carrying an RNA structure, RRE (Rev responsive element) which is located in the env coding region of the genome. Rev interacts with the RRE as a multimer through a basic arginine rich region present in the amino terminal half of the protein. A consequence of this interaction is the transport of partially spliced RNAs that will provide gene products such as Env, Vif, Vpu and Vpr as well as unspliced RNAs that will serve as new genomes for incorporation into assembling particles. Structural analysis of the Rev/RRE interaction has also been carried out using NMR (Battiste et al. (1996) Science, 273:1547–1551.1996). Rev carries a leucine rich export signal that allows it to shuttle between the nucleus and the cytoplasm for continued transport of newly synthesised RNAs (Kalland et al. (1994). J. Virol., 68:1475–1485; Meyer & Malim, (1994) Genes Dev., 8:1538–1547.). In this way, Rev ensures that the structural genes are expressed late following the regulatory genes. Therapeutic strategies directed against Rev will interrupt the viral life cycle early in infection.
Although originally described as a negative factor, Nef has later been shown to have positive effects on virus replication and is expressed in larger quantities than that of Tat and Rev, both early and throughout infection. Nef is myristylated at the N-terminus and is associated with the inner side of the plasma membrane. Nef is partly responsible for down regulation and degradation of surface CD4 by endocytosis (Piguet et al. (1998) EMBO J., 17:2472–2481.). Removing CD4 from the cell surface prevents superinfection with other HIV-1 strains, or reinfection with newly released virus. Nef is also responsible for the down regulation of MHC class I thereby protecting infected cells from destruction by cytotoxic T-lymphocytes (Le Gall et al. (1997). Res. Virol., 148:43–47.). Nef is not an essential viral protein since it is not required for in vitro infection of peripheral blood lymphocytes or T-cell lines. Nef deletion mutants, however, are less pathogenic over long periods of time. Nef also has complex effects on signal transduction pathways in the cell and contains a proline rich region that can interact with the SH3 domain of kinases involved in T-cell activation, a feature necessary for efficient HIV replication (Moarefi et al. (1997). Nature, 385:650–653.). Nef containing viruses are capable of more viral DNA synthesis than viruses deleted in the Nef gene which suggests that Nef directly or indirectly activates the viral reverse transcriptase. The low level of Nef associated with virions may be responsible for this phenomenon. Low levels of Nef are also released from infected cells although the potential effect on neighbouring cells is unclear. Since Nef is expressed early in infection and has significant effects on CD4 and MHC class I expression as well as disease progression, it represents an important target for future therapeutic strategies.
Tat and Nef are secreted and can be taken up by macrophages and expressed in association with MHC class II molecules. This improves their suitability as targets for peptide based therapies which also would be expressed in the context of MHC class II. It is clear that targeting early gene products that are essential for HIV-1 replication, such as Tat and Rev should be given priority in addition to Nef which is also expressed early and influences disease progression.
TABLE 1HIV-1 Regulatory and auxiliary proteins.GeneProteinNameExpressionLocalisationFunctionsTatTatTransactivatorEarlyNucleusActivates viralof viraltranscription.transcriptionSecreted frominfected cells where itcan activate T-cells,induce apoptosis andfunction as a growthfactor.RevRevNuclear RNAEarlyNucleolus,Regulatesexport factornucleoplasm,splicing/RNAcytoplasmtransport to thecytoplasm. A shuttleprotein.NefNefNumerousEarlyCytoplasm,Triggers CD4effectormembraneendocytosis, Downfunctionsassociated.regulates MHC classVirions1 expression. Bindsto kinases and mayinfluence T-cellsignalling andactivation.VpuVpuViral protein uLateCytoplasm,Triggers intracellularmembraneCD4 degradation,associateddown regulates MHCclass 1, nonspecificpromoter of retroviralparticle release.VifVifViral infectivityLateCytoplasm,Enhances infectivityfactormembranesof viral particles in aVirionscell dependentmanner. Improvesviral DNA synthesisduring reversetranscription.VprVprViral protein rLatePredominantlyContributes tonucleusnuclear import ofVirionspreintegrationcomplex.Arrests cells in G2/Mphase of the cellcycle.
Naturally occurring HIV sequences in vaccine candidates are not capable of stimulating a stable longterm immune response due to HIVs inherent ability to hide by changing the appearance of the epitopes presented for the immune system. To overcome this variable presentation of epitopes, certain amino acid substitutions and amino acid combinations will support the immune system to present and recognize these foreign virus antigens in a reliable manner and thus to a greater extent.
Based on the above background, we decided to investigate the possibility of designing novel synthetic peptides which can mimic the epitopes from the regulatory and auxiliary HIV proteins in such a way that they can be exposed for both the humoral as well as the cellular part of the immune system, to meet the need for an effective therapeutic and/or prophylactic vaccine.
The initital work was based on the native Tat amino acid sequences published by Korber B., et al., Human Retroviruses and AIDS 1997 Eds.Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, N. Mex. The first Tat epitope is located between amino acid 1 and amino acid 24 of the tat protein:
TABLE 2Tat epitopeAA noNaturally occurring AAs1MS2EDV3SQPLVA4VI5DN6PHA7RNSKED8LIQRVMT9EDP10PS11W12KNEHL13HRQ14P15GP16SNA17QKT18PH19KTSARPEQ20TAI21APDV22CS23TNS24NKRQAPT
The one letter as well as the three letter codes defining the amino acids in the sequences given throughout this specification are in accordance with International standards and given in textbooks, for instance Lehninger A. L., <<Principles of Biochemistry>>, Worth Publishers Inc., New York, 1982. The amino acids given to the right of the left column, represent the natural variation of the sequence. Our analyses resulted in a sequence containing this modified epitope:
C S W V N P R L E P W L H P G S Q P NI T A C T N |__________________________________________|
Wherein NI indicates 2-aminohexanoic acid (Norleucine, abbreviated Nle and NI in the three letter and one letter code, respectively) and the cysteine residues are in an oxidized state, i.e. are forming an intrachain disulphide bridge. Since the Cystein residue to the C-terminal part (in position 22) of the peptide is part of an intramolecular disulfide bond outside this selected epitope, a similar intrapeptide disulphide bond is formed by placing a Cystein in the N-terminal part of the selected epitope. Another alternative is to form an intermolecular disulphide bond by dimerization of the sequences:
W V N P R L E P W L H P G S Q P NI T A C T N                                        | W V N P R L E P W L H P G S Q P NI T A C T N
A further alternative is dimerization with another epitope selected from Tat. The second epitope on Tat is located between amino acid 35 and 57 in C-terminal direction, separated from the first epitope by 10 amino acids containing 5 Cys residues in addition to the Cys residue in each of the epitopes. The relatively high number of Cys residues offers a variety of inter- and intramolecular crosslinking possibilities. It is likely that this Cys-rich domain will dominate the immunological exposure of this protein and hence cause a “hiding” of the two relevant epitopes. Selection and modification of the two adjacent epitopes can expose an essential part of the Tat protein in a more optimal way.
In order to reduce the probability for development of escape mutants, the number of epitopes is further increased and two additional peptide sequences were selected. These sequences are located on Rev (residues 58–78) and Nef (residues 65–85). The native sequences have been published in Human Retroviruses and AIDS 1999; A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Eds.Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos:
TABLE 3Tat epitopeAA no.Naturally occurring AAs35QPILTYV36VACL37C38F39ILQMT40TNKR41KQ42GA43L44GS45I46SFY47YN48G49RKS50K51K52R53RKSG54QRP55R56R57RGS
TABLE 4Rev epitope:AA no.Naturally occurring AAs58RWQ59IVLF60LIP61GSNDCVTAR62TADNS63YCFRHSVL64LV65GH66RG67SPFL68ATEQVPS69EKQDN70PSAN71VNGP72PQHSLRTDI73LFV74QLPHDE75L76P77PLE78LIVPand the
TABLE 5Nef epitope, Table 5:AA no.Naturally occurring AAs65EGDS66NGDE67EV68AG69LF70P71IV72TRKAM73P74QH75VLI76P77LVT78R79P80MVI81TD82YFR83KR84AGSEQ85ASV
Several modified peptides have been synthesized in order to determine the uniqueness of the sequences as well as their properties for stimulation of the immune system in combination with their specificity and sensitivity as HIV-1 antigens.